Meat Science 96 (2014) 526–534

Contents lists available at ScienceDirect

Meat Science
journal homepage: www.elsevier.com/locate/meatsci

Influence of natural extracts on the shelf life of modified

atmosphere-packaged pork patties
José M. Lorenzo a, Jorge Sineiro b, Isabel R. Amado c, Daniel Franco a,⁎
a
Centro Tecnológico de la Carne de Galicia, Rúa Galicia Nº 4, Parque Tecnológico de Galicia, San Cibrao das Viñas, 32900 Ourense, Spain
b
Department of Chemical Engineering, School of Engineering, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain
c
Grupo de Reciclado y Valorización de Materiales Residuales (REVAL), Instituto de Investigaciones Marinas (CSIC), 36208 Vigo, Spain

a r t i c l e i n f o a b s t r a c t

Article history: In this study four natural extracts from tea (TEA), grape (GRA), chestnut (CHE) and seaweed (SEA) with potential
Received 6 March 2013 antioxidant activity were evaluated in pork patties. During 20 days of storage in modified atmosphere packs at
Received in revised form 31 July 2013 2 °C, pH, colour, lipid oxidation and microbial spoilage parameters of raw minced porcine patties were examined
Accepted 7 August 2013
and compared with a synthetic antioxidant (BHT) and control (CON) batch. Due to their higher polyphenol con-
tent, GRA and TEA extracts were the most effective antioxidants against lipid oxidation, also limiting colour de-
Keywords:
Display life
terioration. In addition, both natural extracts led to a decrease of total viable counts (TVC), lactic acid bacteria
Microbial spoilage (LAB), Pseudomonas and psychotropic aerobic bacteria compared to the control. Among the four natural com-
Natural antioxidant pounds tested, tea and grape extracts showed the most potential as alternatives to commercial antioxidants,
Oxidative stability for increasing the quality and extending the shelf-life of porcine patties.
© 2013 Elsevier Ltd. All rights reserved.

1. Introduction Antioxidants are added to meat products during processing to delay

Microbial growth, lipid oxidation and colour are important
factors to shelf-life and consequently for the consumer acceptance
of fresh meat. Oxidative processes in meat and meat products during
storage lead to the degradation of colour pigments, lipids and
proteins that, in turn, can contribute to the deterioration in flavour,
texture, colour and nutritional value of meat (Rodríguez-Carpena,
Morcuende, & Estévez, 2011).
Lipid oxidation is a critical point for meat packaged under aerobic
conditions, limiting their quality and acceptability since it affects the
sensory properties, due to off-flavour and off-odour development, and
the production of potentially toxic compounds such as fatty acid perox-
ides, cholesterol hydroperoxide, and peroxy radicals (Jakobsen &
Bertelsen, 2000). In addition, colour changes are an important factor
influencing the quality and acceptability of meat and meat products.
Colour tends to be used as an indicator of quality and freshness of
meat, since Carpenter, Cornforth, and Whittier (2001) confirmed that
there is a close relationship between colour preference and the decision
to purchase. Storage of meat in high-oxygen atmospheres, such as
modified atmosphere packaging with up to 80% oxygen, is useful for
colour protection but has been found to degrade meat quality
(Clausen, Jakobsen, Ertbjerg, & Madsen, 2009; Lund, Lametsch, Hviid,
Jensen, & Skibsted, 2007).
⁎ Corresponding author. Tel.: +34 988 548 277; fax: +34 988 548 276.
E-mail address: danielfranco@ceteca.net (D. Franco).
oxidation (Djenane, Sánchez-Escalante, Beltrán, & Roncalés, 2002). In
an attempt to control this process, food industries use synthetic antiox-
idants such as butylated hydroxyanisole (BHA), butylated- hydro
xytoluene (BHT), propyl gallate (PG) and tert-butylhydroquinone
(TBHQ). However, these products may exhibit toxic properties in the
humans as they are suspected to be carcinogenic (Madhavi &
Salunkhe, 1995) and thus counteract any beneficial effect for the
consumer (Tamil Selvi, Joseph, & Jayaprakasha, 2003). This is one of
the reasons for increased demand for “natural food ingredients” of
plant origin (Rojas & Brewer, 2008). Agro-industries generate numerous
waste products resulting in environmental problems and therefore
the achievement of a sustainable agriculture implies the reduction/
elimination of waste. The possibility of using these wastes as natural
antioxidants in the food industry could represent a significant step
towards maintaining environmental balance. For instance in wineries,
where wastes account for approximately 30% of the total volume of
grapes used, waste represent serious storage, processing or disposal
problems (Rockenbach, Silva, Rodrigues, Kuskoski, & Fett, 2008).
Among natural antioxidants, extracts rich in phenolic compounds like
those derived from the wine industries have been reported as good
alternatives since they are readily available as industrial wastes and
maintain a potential preservative effect (Baydar, Ozkan, & Sagdic,
2004). Many positive effects on human health have been described for
polyphenols including anti-inflammatory, anti-carcinogenic, cardio-
protective and vasodilatory properties (Bonilla, Mayen, Merida, &
Medina, 1999).
Also, due to the loss of effectiveness of antibiotics caused by the
evolution of pathogen resistance, natural products have been

0309-1740/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.meatsci.2013.08.007
 J.M. Lorenzo et al. / Meat Science 96 (2014) 526–534
investigated as antimicrobials. The products derived from plants, specif-
ically essential oils, contain active ingredients that may act as antimicro-
bial compounds against bacteria, yeast, and molds (Juneja, Dwivedi, &
Yan, 2012). Active compounds in plant-derived antimicrobials include
a wide range of molecules, the most important being polyphenols
(including flavonoids and lignans), quinones and alkaloids (Saleem
et al., 2010).
Many vegetable extracts have been investigated in vitro as natural
sources of these active molecules, including grape seeds, tea, chestnut
and seaweed. For instance, antimicrobial activity of grape seed extracts
has been tested against Bacillus cereus, Bacillus coagulans, Bacillus
subtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas
aeruginosa (Jayaprakasha, Selvi, & Sakariah, 2003). Gallic acid has been
identified as the active compound against E. coli and Salmonella
enteritidis in methanolic grape seeds extracts (Tesaki et al., 1999). Bio-
active compounds from green and black teas are also potent inhibitors
of S. aureus and E. coli (An et al., 2004). Also the antimicrobial activity
of chestnut extracts has been investigated, the active fraction being
rich in flavonol glycoside and terpenoid substances (Hao et al., 2012).
The antimicrobial activity of fourteen seaweed extracts has been
assessed against multiresistant pathogens (Shanmughapriya et al.,
2008), revealing higher inhibitory activity against Gram negative than
Gram positive bacteria, the active compounds being lipophilic.
The aim of this study was to investigate the effectiveness of natural
antioxidants as additives to improve the quality and extend the shelf-
life of meat products. For this purpose, the ability of tea, grape, chestnut
and seaweed extracts to inhibit microbial spoilage, colour deterioration
and lipid oxidation was evaluated in raw porcine patties packaged in
modified atmosphere, during chilled storage.
2. Material and methods
2.1. Grape seed extract (GRA)
Three lots of 2 kg of two seed extracts of varieties of white grape
seed were used (Vitis vinifera “Albariño” variety and a Vitis labrusca
hybrid). The samples were supplied by a local producer (Galicia,
Spain) after being processed for Albariño wine in 2005 and distilled to
obtain spirits. Grape seeds were manually separated, cleaned, washed
and frozen at −50 °C until use. Samples were milled using a laboratory
scale hummer mill (Filtra model FTML-0100, Barcelona, Spain). Samples
were manually sieved to remove the smallest particles using stainless
steel sieves (Filtra, Barcelona, Spain), the final particle size was between
3 and 5 mm. The solvent extract was a mixture of absolute ethanol/
water (ratio 1/1). An extraction pilot-plant was setup as reported by
Sánchez, Franco, Sineiro, Magariños, and Núñez (2009) with the follow-
ing modifications: batches of 2 kg of grape seed were packed into a glass
jacketed column, which was thermostated at 60 °C by water from a
immersion heater (Haake C10, Thermoelectron, Karlsruhe, Germany);
the solvent was pumped upflow through the bed at 113.0 ±
8.2 mL/min, by a peristaltic pump, composed of: a Cole–Parmer
head Easy-load 77601-00 connected to a Baldor three-phase motor
(Fort Smith, Arizona, USA) by an adapter Cole–Parmer 56C 77490-30
and a Zener MSC variable speed drive (Sydney, Australia). The input
flow was preheated by passing the silicon tubes through the water
bed. The extract was collected for 2.5 h.
The extraction yielded around 15.25 L, which were evaporated in a
rotary evaporator (Büchi R-114, Zurich, Switzerland) at 40 °C. The
remaining solid was suspended in 3 L of water. A glass column (7 cm
Øin × 40 cm height) filled with XAD-16 Amberlite was equilibrated
with distillate water. One litre of aqueous suspension extract was
poured into column to obtain purified polyphenolic material. The col-
umn was washed with 5 L of water; later, the polyphenolic material
adsorbed on XAD-16 Amberlite was eluted with ethanol (1 L × 3).
The ethanolic extract was evaporated to dryness and lyophilized using
a Kinetics EZ-Dryer freeze-dryer (FTS Kinetics, Stone Ridge, NY, USA).
527
This raw lyophilized extract was called GRA extract (GRA) and rendered
136.7 ± 34.0 g GRA/kg grape seed.
2.2. Chestnut extract (CHE)
Chestnut (Castanea sativa) leaves (CsL) from the 2005 harvest were
collected in Ribeira Sacra area (Ourense, Spain). CsL were dried at 50 °C
during 3–4 days (depending on the initial moisture content) in an air
oven (Memmert UFP 600, Schwabach, Germany). Air-dried and ground
CsL (final moisture 9.68% and less than 1 mm) were stored in sealed
plastic bags in the dark before use. Aqueous extraction was according
to Díaz-Reinoso, Moure, Domínguez, and Parajó (2011). Briefly, leaves
(1 kg) were extracted with distilled water (25 L) acidified with 0.1 M
HCl under conditions leading to maximal radical scavenging activity
(25 °C during 90 min) in a tank with stirring and temperature control.
Solid:liquid separation was accomplished by vacuum filtration, and
the liquid phase was processed in a series of two ultrafiltration
membranes. Membrane fractionation was performed in an ultrafiltra-
tion (UF) lab pilot home-made plant, consisting of a feed tank of 2.5 L,
a peristaltic Masterflex pump (Cole–Parmer Instrument Co., Chicago,
IL, USA) and a membrane module equipped with 5 and 10 kDa Omega
membranes (Minisette, Pall Filtron, USA) (0.12 mm × 0.10 mm)
having an effective surface area of 0.07 m2. The membrane material
was modified polyethersulfone and the maximum operation pressure
was 4 bar.
2.3. Green tea extract (TEA)
Green tea (Camellia sinensis) leaves were purchased in a local
market, ground, sieved (fractions less than 0.25 mm and greater than
1 mm were rejected) and stored in a dry, dark place at room tempera-
ture before use. Commercial leaves were directly ground in an Ika
Labortechnik mill (Staufen, Germany). The milled green tea leaves
extractions were carried out with 70% ethanol at a liquid–solid ratio
(LSR) of 15 g/g, during 8 h at 30 °C. Extracts were vacuum filtered,
vacuum concentrated up to one third of the initial volume and oven-
dried at 45 °C.
2.4. Seaweed extract (SEA)
Seaweeds (Ulva lactuca and Ulva rigida) were collected during 2009
and 2010 from Vilanova's beaches (Pontevedra, Spain). Seaweeds were
cleaned from sands, stones and small shrimp, washed with running
water, air-dried at 60 °C, crumbled and screened from 0.25 to 2 mm
and stored in a dry and dark place at room temperature before use.
The dried algae were ground in an Ika Labortechnik mill (Staufen,
Germany) at room temperature. Seaweeds were hydrolysed with 1.5%
H2SO4 using an LSR of 60:1 g/g. Hydrolysis was carried out in closed
flasks in an autoclave at 120 °C. After hydrolysis, solid–liquid separation
was accomplished by vacuum filtration and the liquid phase (hydro-
lyzate) was neutralised (pH 6.5) with CaCO3. The aqueous phase was
separated from solids by vacuum filtration and stored in dark bottles
at 4 °C (Cruz, Dominguez, Dominguez, & Parajó, 2001).
Seaweed extracts were obtained from the acid hydrolysis liquors
by ultrafiltration in a membrane home-made module equipped with a
commercial amicon stirred cell (model 8400, Millipore, Billerica, MA,
USA) equipped with a regenerated cellulose membrane (Millipore, Bil-
lerica, MA, USA 1 kDa molecular weight cut-off, 41.8 cm2 effective
area). The equipment consisted of a 10 L feed tank (in which tempera-
ture was controlled by flushing tap water through a refrigeration coil), a
variable speed Hydracell pump, two pressure gauges at the membrane
inlet and outlet to measure the TMP, a needle valve located after the
membrane to achieve the desired TMP, and a flowmeter to measure
the recycle flow. Operation was carried out at room temperature
(20 °C) and the TMP was selected to avoid fouling membrane. The
528 J.M. Lorenzo et al. / Meat Science 96 (2014) 526–534
concentrate was freeze-dried (Christ Alpha 2-4 LDPlus, Osterode am
Harz, Germany).
2.5. Determination of antioxidant capacity
2.5.1. Chemicals
All chemicals and reagents were of analytical grade. Folin–Ciocalteu
reagent, gallic acid, ABTS (2,2-azinobis-(3-ethyl-benzothiazoline-6-
sulfonate)) radical, Trolox, β-carotene, Tween-40, DPPH (α, α-
Diphenyl-β-picrylhydrazyl) radical and BHT (butylated hydroxytoluene)
were from Sigma Aldrich (St. Louis, MO). Linoleic acid was from Fluka
(Steinheim, Germany) and the other chemicals were from Panreac
Química S.A. (Barcelona, Spain).
2.5.2. Determination of total phenolic content
The total phenolic content was determined following the method of
Singleton, Orthofer, and Lamuela-Raventós (1999), using the Folin–
Ciocalteu reagent (FCR) with gallic acid as a standard. Fifty microlitre
of sample or blank were added to 3 mL of distilled water in test tubes.
FCR (250 μL) was placed into the tube and mixed before adding
750 μL of saturated Na2CO3. The final volume of the reaction mixture
was adjusted to 5 mL with distilled water and after incubation for 2 h at
room temperature; the absorbance at 765 nm was read in 1 cm cuvettes
(PerkinElmer® Lambda 25 UV/Vis spectrophotometer, PerkinElmer Inc.,
Massachusetts, USA). Readings were compared with a standard curve of
gallic acid, being the total phenolic content expressed as mg of gallic
acid equivalent per g of freeze dried solid (mg GAE/g).
2.5.3. Trolox equivalent antioxidant capacity (TEAC)
The Trolox equivalent antioxidant capacity was determined fol-
lowing the protocol of Re et al. (1999) with some modifications.
This assay is based on the scavenging of ABTS radical observed as
a decolourization of the blue–green colour at 734 nm. Solutions
of 7 mM ABTS and 2.45 mM potassium persulfate in phosphate
buffer saline (PBS, pH 7.4) were allowed to stand overnight to
generate the ABTS radical cation (ABTS•+). Then, the ABTS•+
stock solution was diluted with PBS and equilibrated at 30 °C to
an absorbance of 0.8–0.9 at 734 nm (PerkinElmer® Lambda 25
UV/Vis spectrophotometer, PerkinElmer Inc., Massachusetts, USA).
Triplicate determinations were performed by mixing 100 μL of the sam-
ple with 1.9 mL of radical solution. The decline in absorbance was
followed for 10 min. Appropriate solvent blanks were run for each sam-
ple. The radical scavenging capacity was compared with that of Trolox
and results were expressed as g of Trolox equivalent per g of freeze
dried solid.
2.5.4. β-Carotene bleaching assay
The β-carotene (βC) bleaching assay described by Marco (1968)
was modified for use with microplates. Briefly, 4 mg of βC, 0.5 mL
of linoleic acid and 4 g of Tween-40 were dissolved in 20 mL of chlo-
roform, the stock solution was distributed in aliquots of 1 mL and the
chloroform was evaporated in a rotary evaporator (model R-210,
Buchi, Flawil, Switzerland) at 50 °C for 15 min. To remove oxygen,
the tubes containing the resulting oily residue were flushed with
nitrogen and stored at − 18 °C. For each microplate, 1 mL of the
stock solution was resuspended in 25 mL of milli-Q water at the assay
temperature (45 °C). The absorbance at 470 nm of the reagent thus pre-
pared was ~1.2.
For measuring the antioxidant capacity 50 μL of sample were mixed
with 250 μL of reagent in a 96 well microplate. Samples were analysed
in triplicate, in a concentration range between 50 and 500 mg/L (final
concentrations). Also appropriate solvent blanks were run for each
sample. A series in which the sample was replaced by a commercial
antioxidant (BHT) at the appropriate concentrations (0.5–5 mM) in
the reaction mixture was included in each microplate.
Absorbance readings (470 nm) were taken at regular intervals
in a ThermoFisher Scientific microplate reader (Waltham, MA,
USA) until β-carotene was decoloured (about 3 h). The antioxidant
activity coefficient (AAC) was calculated as follows:
 
Asample −ðAcontrol Þt¼120
AAC ¼ t¼120
 1000 ½1
ðAcontrol Þt¼0 −ðAcontrol Þt¼120
where Asample is the absorbance at 470 nm of the β-carotene in the
presence of sample and Acontrol is the absorbance at 470 nm of the
β-carotene in its absence.
The plot of AAC versus concentration of antioxidant generates dose–
response curves can be described by a sigmoidal model defined by the
Weibull equation modified to have the EC50 (Rial, Vázquez, & Murado,
2011):
   a 
C
A ¼ K 1− exp − ln2 ½2
m
where, A is antioxidant capacity (dimensionless), K is the maximum
activity (dimensionless), C is the concentration of antioxidant (g/L),
m is the concentration for semi-maximum response (EC50 in g/L) and
a is a parameter related to the maximum slope of the function
(dimensionless).
The activity was also expressed as BHT equivalent activity (g BHT/g
freeze dried solid), being the equivalent BHT concentration determined
from the calibration curve obtained by plotting AAC versus BHT
concentrations.
2.5.5. α, α-Diphenyl-β-picrylhydrazyl (DPPH) radical scavenging activity
The antioxidant activity was determined with DPPH as a free
radical, using an adaptation of the method described by Brand-
Williams, Cuvelier, and Berset (1995). For the modified procedure,
antioxidant solutions (10 μL) were added in triplicate to 200 μL of a
60 μM solution of DPPH• in 70% ethanol. The decrease in absorbance
was followed at 515 nm every 5 min until the reaction reached a pla-
teau (about 2 h). The EC50 and BHT equivalent activity were calculated
as explained above.
2.6. Preparation of samples and package conditions
Six batches of ground meat patties [control (C), BHT, GRA, CHE, TEA
and SEA] were manufactured. Pork patties of 100 g (n = 3 per batch
and storage time) were manufactured using the primal cuts of pig
shoulder and loin. These primal cuts, with a total fat content of 8.75%
were ground using a 6 mm plate in a refrigerated mincer machine
(La Minerva, Bologna, Italy). The meat was mixed and compressed by
hand with 10 g of NaCl per kg of meat and 50 mg/kg of BHT or
1000 mg/kg of each natural extract.
Meat mass mixed was maintained under refrigeration for 20 h and,
after this time, pork patties were produced in moulds of 10 cm diameter
and 1 cm height in a burger-maker (Gaser, A-2000, Girona, Spain). Pork
patties were packed in 300 mm thick polystyrene trays, which were
sealed with polyethylene film 74-mm thick and permeability b 2 mL/
(m2 bar day) suitable for gas mixtures (VIDUCA, Alicante, Spain). The
packaging was carried out using a heat sealer LARI3/Pn T-VG-R-SKIN
(Ca.Ve.Co., Palazzolo, Italy). The composition of the modified atmo-
sphere was 80% O2–20% CO2. The trays were stored at 2 ± 1 °C with
light, to simulate supermarket conditions. The trays were placed over
metal shelving and receiving lux values (digital luxometer, HT 306,
Italy) in the range of 15–20 depending on the tray position. The light
source was conventional, so any wavelength or range (for instance
UV) was not filtered. Analyses were carried out at 1, 6, 13 and 20 days
of storage. The pH, colour, lipid oxidation, and microbial spoilage values
were determined in duplicate for every sampling point.
 J.M. Lorenzo et al. / Meat Science 96 (2014) 526–534
2.7. Microbial analysis
Samples of 10 g of patties were aseptically weighed and homo-
genised with 90 mL of 0.1% sterile peptone water in a masticator blend-
er (IUL Instruments, Barcelona, Spain) for 2 min at room temperature.
For each sample, serial decimal dilutions were prepared in 0.1% peptone
solutions (Merck, Darmstadt, Germany), and duplicate 1 mL or 0.1 mL
samples of the appropriate dilutions were poured or spread onto total
count and selective agar plates, respectively.
Total viable counts (TVC) and psychotropic aerobic bacteria were enu-
merated in Plate Count Agar (PCA; Oxoid, Unipath Ltd., Basingstoke, UK)
after incubation at 30 °C for 48 h and 7 °C for 10 days, respectively. Lactic
acid bacteria were determined on the Man–Rogosa–Sharpe medium Agar
(Oxoid, Unipath Ltd., Basingstoke, UK) at pH 5.6, after incubation at 30 °C
for 5 days. Pseudomonas spp. were counted on Pseudomonads Selective
Agar (Merck, Darmstadt, Germany) with Pseudomonads CFC Selective
Supplement (Merck, Darmstadt, Germany) and incubated at 25 °C for
48 h. After incubation, plates with 30–300 colonies were counted.
Microbiological data were transformed into logarithms of the number of
colony forming units (CFU/g).
2.8. pH and colour parameters
pH was measured using a pH-meter (Hanna Instrument HI-9024,
Portugal) equipped with a glass probe for penetration. A portable color-
imeter (Minolta CR-600d Osaka, Japan) with the following settings:
pulsed xenon arc lamp, 0° viewing angle geometry and aperture
size of 8 mm, was used to measure meat colour in the CIELAB space
(Lightness, L*; redness, a*; yellowness, b*) (CIE, 1978) and MetMb
percentage of the total surface myoglobin. The relative content of
metmyoglobin at the surface of the loin is based on measurements of re-
flex attenuance of incident light at 572, 525, 473 and 730 nm
(Krzywicki, 1979). Colour measurements were made on the surface of
each patty in triplicate at three randomly selected locations. The colour
difference (ΔE) between patties at day 1 and day 20 of storage was cal-
culated as: ΔE1 − 20 = [(L20 − L1)2 + (a20 − a1)2 + (b20 − b1)2]1/2.
2.9. Lipid oxidation
Lipid stability was evaluated as proposed by Vyncke (1975). Briefly,
2 g of meat was dispersed in 5% trichloroacetic acid (10 mL, Scharlau,
Sentmenat, Barcelona, Spain) and homogenised in an Ultra-Turrax (Ika
T25 basic, Staufen, Germany) for 2 min. The homogenate was
maintained at −10 °C for 10 min and centrifuged (Beckman Coulter
Allegra X-22, Fullerton, California, USA) at 2360 ×g for 10 min. The su-
pernatant was filtered through a Whatman No. 1 filter paper (Maidstone,
UK). The filtrate (5 mL) was reacted with 0.02 M TBA solution (5 mL,
Acros Organics, Geel, Belgium) and incubated in a water bath at 96 °C
for 40 min. The absorbance was measured at 532 nm. Thiobarbituric
acid reactive substances (TBAR'S) values were calculated from a standard
curve of malonaldehyde with 1,1-3,3 tetraethoxypropane (TEP) and
expressed as mg MDA/kg sample.
2.10. Statistical analysis
An analysis of variance (ANOVA) using the General Linear Model
(GLM) procedure of the SPSS package (SPSS 19.0, Chicago, IL, USA)
was performed for all variables in the study. When the effect of both
natural antioxidants and time of storage on microbial analysis,
physico-chemical traits and lipid oxidation were studied their fixed
effects were included in the model. The model used was:
Yij ¼ μ þ Si þ A j þ εij
where:Yij is the observation of dependent variables, μ is the overall
mean, Si is the effect of display days, Aj is the effect of natural
529
antioxidants, and εij is the residual random error associated with the ob-
servation. Correlations between variables (P b 0.05) were determined
by correlation analyses using Pearson's linear correlation coefficient
with above statistical software package.
3. Results and discussion
3.1. In vitro antioxidant activity of the extracts
The antioxidant activities of TEA, GRA, CHE and SEA extracts are
shown in Table 1. The highest polyphenol contents were in GRA and
TEA extracts. The fact that these plant extracts showed greater phenolic
contents is expected, since both green tea and grape seed extracts are
known for their high contents of polyphenolic and proanthocyanidin
compounds (Perumalla & Hettiarachchy, 2011). Polyphenols in these
extracts consist mainly of flavonoids, flavan-3-ols being the major com-
pounds. Monomers, catechin and epicatechin together with cinnamic
acids and sugar-linked flavonols (quercetin, myricetin and kaempferol)
are the major compounds in tea extracts (van der Hooft et al., 2012) and
benzoic acids (gallic and protocatechuic acid), monomer flavan-3-ols
and oligomeric procyanidins are major compounds in grape seed
extract (Rubilar, Pinelo, Shene, Sineiro, & Nuñez, 2007). The antioxidant
activity of flavan-3-ols and flavonols has been demonstrated
(Jayaprakasha et al., 2003; Rubilar et al., 2007). Also, the high activity
of grape seed extract could be due to their resveratrol content, a
known free radical scavenger (Fontecave, Lepoivre, Elleingand, Gerez,
& Guittet, 1998).
The concentrations of soluble polyphenols in macroalgae and chestnut
extracts were an order of magnitude lower. As can be seen in Table 1, the
polyphenol contents in both extracts ranged from 8 to 25 mg GAE/g as
reported for different seaweed varieties (López-López et al., 2009) and
slightly lower than the 103 mg GAE/g found in leaf chestnut extracts
(Barreira, Ferreira, Oliveira, & Pereira, 2008).
The antioxidant activities assessed by the three in vitro methods
(TEAC, DPPH and β-carotene) were directly related to the concentration
of polyphenols in the extracts, the highest activities being in grape and
tea extracts (Table 1). Regarding TEAC activity, the GRA and TEA
extracts had a 10-fold and nearly 50-fold higher Trolox equivalent anti-
oxidant capacities than the CHE and SEA extracts, respectively.
Similarly, the extracts with the higher phenolic contents also pro-
duced greater scavenging activity on DPPH radicals. GRA and TEA equiv-
alent activities were almost twice the antioxidant power of BHT and
almost 4-fold and 4.5-fold higher than that of CHE extracts (Table 1).
SEA extract showed the lowest DPPH activity, about four times less
active than BHT.
The EC50 values obtained for the scavenging effects on DPPH re-
vealed a very powerful antioxidant activity of GRA and TEA extracts
(less than 0.2 g/L). In fact, GRA extract EC50 value (160 μg/mL) was
higher than reported for seeds of red grape (V. vinifera L.) varieties,
which ranged between 2.71 and 4.62 μg/mL (Bozan, Tosun, & Özcan,
2008). On the contrary, similar EC50 values were found in ethanolic
extracts of Italian grape seed extracts (Mandic, Dilas, Ćetković,
Čanadanović-Brunet, & Tumbas, 2008). On the other hand, radical-
scavenging activity did not show a sigmoidal profile with increasing
concentrations of CHE (data not shown) and therefore the EC50 param-
eter on the Weibull model was non-significant (P N 0.05). Similarly, the
graphical plot of DPPH activity vs. algae extract concentration did not
show a clear trend (Table 1).
β-Carotene bleaching results revealed similar activity values for all
extracts, however GRA and TEA were the most active (Table 1). Never-
theless, better EC50 values than those obtained for scavenging on DPPH
radical were observed, with activities of 100 μg/mL for GRA and TEA ex-
tracts. 2-Fold and 4-fold higher EC50 values were calculated for CHE and
SEA extracts, respectively. Chestnut EC50 values were lower than those
obtained by Barreira et al. (2008) for chestnut flower, outer and inner
skin and fruits.
530 J.M. Lorenzo et al. / Meat Science 96 (2014) 526–534

Table 1
Total polyphenol contents expressed as gallic acid equivalents (GAE) and in vitro antioxidant activity determined by the Trolox equivalent antioxidant capacity (TEAC), α, α-Diphenyl-β-
picrylhydrazyl (DPPH) radical scavenging method and β-carotene (βC) bleaching assay of four natural antioxidants. Both BHT equivalent activity and EC50 value are shown for each extract
and method used.

Total polyphenols (mg GAE/g extract) TEAC (g Trolox/g extract) DPPH β-carotene

EC50 (g extract/L) ratio (g Eq BHT/g extract) EC50 (g extract/L) Ratio (g Eq BHT/g extract)

GRA 373.0 ± 6.1 2.93 ± 0.03 0.16 ± 0.02 1.80 ± 0.28 0.05 ± 0.01 1.28 ± 0.22
CHE 89.0 ± 0.3 0.27 ± 0.02 1.04 (NS) 0.48 ± 0.11 0.10 ± 0.01 0.53 ± 0.11
TEA 390.9 ± 8.6 4.06 ± 0.28 0.12 ± 0.01 2.18 ± 0.12 0.05 ± 0.01 0.69 ± 0.19
SEA 12.8 ± 1.7 0.06 ± b0.01 – 0.24 ± 0.05 0.23 (NS) 0.26 ± 0.09

GRA: Grape seed; CHE: Chestnut; TEA, Tea and SEA: Seaweed.

Overall, GRA and TEA extracts showed the highest polyphenol con-
tents and antioxidant activity as determined by TEAC, β-carotene
bleaching assay and scavenging of DPPH radical. GRA and TEA extracts
showed a BHT equivalence higher than 1 for DPPH assay and GRA ex-
tract and for β-carotene assay. TEA extract showed a β-carotene protec-
tion capacity slightly lower than BHT (0.69 g/g). Since in this assay the
medium is an emulsified biphasic system, the ability to act in the inter-
phase is critical and perhaps not all antioxidant polyphenols are ade-
quately distributed in the vicinity of the interphase to act as effectively
as BHT. Several authors have reported on the effect of degree of poly-
merization (Tourino et al., 2005) or chain length (Lotito et al., 2000)
on the antioxidant activity by in vitro methods.
3.2. Evolution of microbial counts during storage of porcine patties
Changes in total viable counts (TVC) are shown in Fig. 1A. In general,
significant differences (P b 0.05) were observed in microbial counts dur-
ing storage and among treatments. Initial TVC (day 1) were around
4 log10 CFU/g, except for SEA group that showed the highest values
(5.1 log10 CFU/g). After 6 days of storage, TVC increased significantly
(P b 0.001) until the end of the refrigerated period, when patties from
TEA, GRA and BHT presented lower TVC than the control, while patties
from SEA and CHE showed the highest levels (above 8 log10 CFU/g).
Thus TVC decreased significantly (P b 0.001) with the addition of tea
and grape extracts, while seaweed and chestnut extract did not signifi-
cantly affect microbial growth. These results are in agreement with
those of An et al. (2004) and Jayaprakasha et al. (2003), who observed
an in vitro antibacterial effect of grape and tea extracts. According to Span-
ish legislation (Regulation EC 2073/05, DOUE L338/1, 2005) the limit for
bacterial counts is 106 CFU/g, as spoilage can be detected, mainly due to
odour, in most foods with more than 6 log10 CFU/g (Dainty & Mackey,
1992). According to this criteria, the shelf-life of control patties would
be 13 days, while TEA patties could extend this shelf-life to 20 days.
Lactic acid bacteria (LAB) constitute a substantial part of the natural
microflora of MAP meats (Chouliara, Karatapanis, Savvaidis, &
Kontominas, 2007). At the beginning of storage, lower LAB counts
were observed in treated patties (below 4 log10 CFU/g) than in control
patties (Fig. 1B). However, during the whole storage period, LAB counts
increased significantly (P b 0.001), reaching lower values in TEA and
GRA patties (below 7 log10 CFU/g) than in CHE and SEA patties (above
8.5 log10 CFU/g) after 20 days. Thus addition of tea and grape extracts
reduced LAB growth in porcine patties.

A B

C D

Fig. 1. Changes in microbial counts of porcine patties packed in modified atmosphere and stored at 2 ± 1 °C as affected by natural antioxidants. A: total viable counts (TVC), B: lactic acid
bacteria (LAB), C: Pseudomonas spp. and D: psychrotrophic aerobic bacteria counts. Symbols: , control; , chestnut; , seaweed; , BHT; , grape seed, and , tea extracts.
 J.M. Lorenzo et al. / Meat Science 96 (2014) 526–534 531

Pseudomonas have been characterised as the organisms responsible of (6.66), due to the nature of their polyphenols, phlorotannins, which
the spoilage of aerobically stored fresh meat (Koutsoumanis, Stamatiou, do not contain acidic groups. On the other hand, pH values of CHE and
Skandamis, & Nychas, 2006). As can be seen in Fig. 1C, differences TEA extracts ranged from 4.57 to 5.74, explaining the statistically signif-
(P b 0.05) among treatments were observed in Pseudomonas counts icant differences observed in pH values of porcine patties.
after 1 day of storage (Fig. 1C), patties containing CHE and SEA extracts
had lower (P b 0.05) counts than the other treatments. Pseudomonads 3.4. Evolution of colour parameters during storage of porcine patties
are Gram negative bacteria sensitive to CO2 (Jay, Loessner, & Golden,
2005). Thus, CO2-enriched atmospheres such as low O2 MAP, inhibits Instrumental colour parameters measured at the surface of the pork
the growth of Pseudomonads, when compared to overwrap packaging patties during 20 days of refrigerated storage are shown in Fig. 3. Patty
(Lorenzo & Gómez, 2012). Different evolutions among groups were colour characteristics presented significant differences (P b 0.05) among
observed during chilled storage. For instance, patties treated with BHT, treatments and storage time. The addition of natural antioxidant extracts
TEA and GRA showed a continual decrease in the Pseudomonas population had a significant effect on the colour surface displayed by freshly made
during storage whereas in SEA and CHE patties a significant (P b 0.001) patties (day 1). TEA and SEA extracts led to patties with significantly
increase was observed. (P b 0.001) lower a* values, while CHE and GRA extracts caused a signif-
Finally, it was observed that both the addition of natural extracts and icant (P b 0.001) increase of b* values. Lightness was not influenced by
storage time had a significant (P b 0.05) effect on the psychotropic aer- the addition of natural antioxidant extract.
obic bacteria counts (Fig. 1D). Initial psychotropic aerobic bacteria
counts (day 1) were below 4 log10 CFU/g, except SEA and control
patties that showed higher values (4.5 log10 CFU/g). All patties showed
a significant (P b 0.05) increase in psychotropic aerobic bacterial
A
counts during refrigerated storage. At the end of storage, TEA and GRA
patties presented lower psychotropic aerobic bacterial counts (5.6
and 6.5 log10 CFU/g, respectively) than control and BHT patties
(7.7 log10 CFU/g). CHE and SEA patties showed the highest levels,
being both above 8 log10 CFU/g. The results obtained using TEA and
GRA extracts, are in agreement with those reported by Djenane et al.
(2002), who detected an inhibitory effect of rosemary extract on
psychrotrophic growth when applied to the surface of beef steaks.

3.3. Evolution of pH during storage of porcine patties

The pH levels did not show significant (P N 0.05) differences during

storage, reaching an average value below 6.10 after 20 days. Neverthe-
less, statistical analysis indicated that the addition of antioxidants
B
affected patties' pH values (P b 0.001) leading to lower pH values in
TEA, BHT and GRA patties than in CON, CHE and SEA ones throughout
storage (Fig. 2). These results could be explained by the properties of
active compounds in these extracts. In fact, grape seed containing gallic
acid, protocatechuic acid and galloylated proanthocyanidins was the
most acid extract (4.29) while seaweed had the highest pH value

Fig. 3. Changes in colour parameters in the CIELAB space of porcine patties packed in mod-
Fig. 2. pH evolution of porcine patties packed in modified atmosphere during storage at ified atmosphere during chilled storage at 2 ± 1 °C as affected by natural antioxidants. A:
2 ± 1 °C, as affected by natural antioxidants. , control; , chestnut; , seaweed; , L* values, B: b* values and C: a* values. Symbols: , control; , chestnut; , seaweed; ,
BHT; , grape seed, and , tea extracts. BHT; , grape seed, and , tea extracts.
532 J.M. Lorenzo et al. / Meat Science 96 (2014) 526–534
L* values gradually decreased over time in control and TEA, while the
other groups exhibited an increase with storage time (Fig. 3A). All types
of patties showed a significant (P b 0.05) decrease in redness during
refrigeration, L* values being significantly higher (P b 0.001) in CHE
and BHT groups than in the rest of treatments. A slight decrease of
yellowness was observed in control patties (Δb*: −0.55), while an in-
crease was detected in the treated groups (Δb* ranged from 0.09 to
1.86) (Fig. 3B).
These results confirm that patties suffered discolouration, mainly
defined by loss of redness, and are in agreement with those reported
by other authors (Estévez, Morcuende, & Cava, 2003; Haak, Raes, & De
Smet, 2009; Rodríguez-Carpena et al., 2011) in raw meat during chilled
storage. The final a* values indicate a brown colour since a* values rang-
ing from 3.2 to 4.6 are perceived as grey while values ranging from 4.6 to
10.8 are perceived as brown in pork meat (Santos, Rojas, Lockhorn, &
Brewer, 2007). In the present work, the addition of grape extract
resulted in higher a* values compared to the control and the other treat-
ments (Fig. 3C). This stabilising effect of natural antioxidants on colour
has been observed in studies with other natural antioxidant such as
avocado (Rodríguez-Carpena et al., 2011), rosemary and lemon balm
(Lara, Gutierrez, Timón, & Andrés, 2011), carnosine (Badr, 2007) and
red grape (Garrido, Auqui, Martí, & Linares, 2011).
The discolouration that occurs during storage of red meat cuts is
generally associated with oxidation of the iron atom within the heme
group in red oxymyoglobin (OxyMb) to brownish metmyoglobin
(MetMb) (Faustman, Sun, Mancini, & Suman, 2010). The accumulation
of MetMb in the meat surface and subsequent discolouration largely
depends on the presence of reducing systems and on lipid oxidation
(Estévez et al., 2003; Faustman et al., 2010). As can be seen in Fig. 4A,
in all treatments metmyoglobin percentage increased rapidly during
the first 6 days of storage, being negatively correlated to redness (r =
−0.590, P b 0.01). The results showed that TEA and CHE extracts accel-
erated the accumulation of metmyoglobin in pork patties after 20 days
of storage. Within this period, both treatments lead to MetMb percent-
ages of 40.6% and 50.7%, respectively. It has been reported that values
above 40% metmyoglobin cause meat rejection by consumers (Greene,
Hsin, & Zipser, 1971), it should be taken into account when applying
tea and chestnut extracts in meat processing. In contrast, seaweed
extracts were effective in delaying metmyoglobin formation compared
to the control, showing the lowest percentages after 20 days of chilled
storage (28.3%).
Several authors have suggested that colour deterioration during
storage could be explained by the oxidative degradation of certain nitro-
sopigments of raw meats (Bekhit, Geesink, Ilian, Morton, & Bickerstaffe,
2003; Haak et al., 2009). On the other hand, protein radicals and other
Fig. 4. Changes in the percentage of surface metmyoglobin (A) and TBAR'S values (B) of porcine patties packed in modified atmosphere and stored at 2 ± 1 °C, as affected by natural an-
tioxidants. Symbols: , control; , chestnut; , seaweed; , BHT; , grape seed, and , tea extracts.
protein oxidation products are known to induce lipid oxidation
(Viljanen, Kylli, Kivikari, & Heinonen, 2004) and hence, heme pigments
in raw meat could also be affected by their pro-oxidant action.
The positive effect observed from tea extracts on the colour of refrig-
erated patties is supported by the colour differences (ΔE1 − 20). As can
be seen in Table 2, patties with added tea extract showed the lowest
ΔE1 − 20 values (1.90), which is below the value of 2, considered a
limit to appreciate visual changes in colour (Francis & Clydesdale,
1975). This result indicates that consumers will not perceive colour
changes suffered by tea patties during chilled storage. Patties treated
with tea extracts were lighter and less red than control patties. On the
other hand, patties with grape extracts were brighter, redder and
more yellow than control patties. Hence, the addition of grape extracts
on pork patties under MAP and refrigerated conditions would help
preserve the colour of freshly made patties for a longer time during
chilled storage. This protective effect towards the desirable red colour
of raw patties may influence consumers purchase decision.
3.5. Evolution of lipid oxidation during storage of porcine patties
The oxidative stability of the six batches was evaluated throughout
storage by determining the 2-thiobarbituric acid-reactive substances
(TBAR'S) index (Fig. 4B). Statistical analysis indicated that TBAR'S
were significantly affected (P b 0.05) by the storage time and addition
of antioxidants. During this time, TBAR'S increased significantly
(P b 0.001; from 0.09 mg to 0.63 mg MDA/kg) in control patties while
BHT and GRA groups only displayed slight changes. TBAR'S values
were lower in patties treated with BHT, TEA, SEA and GRA extracts
than in the control group. However, patties treated with chestnut
extracts showed the highest values throughout storage (Fig. 4B). Statis-
tical analysis showed that the addition of antioxidant decreased
(P b 0.001) the TBAR'S values, which were in the following order at
the end of storage: CHE N CON N SEA N TEA N BHT N GRA. These re-
sults indicated that grape extract was the most effective against TBAR'S
formation, even better than the commercial antioxidant BHT. Accord-
ingly, the quality of meat patties could be improved by the addition of
grape extract, given that lipid oxidation leads to a loss of sensory and
nutritional properties. Results are in good agreement with these state-
ments, since redness was negatively correlated to TBAR'S values
(r = −0.495, P b 0.01). This correlation suggests that likely interaction
between lipid and myoglobin oxidation.
Georgantelis, Blekas, Katikou, Ambrosiadis, and Dimitiros (2007)
noticed that rancid flavour is detected in meat products with TBAR'S
values higher than 0.6 mg MDA/kg. TBAR'S values from GRA, BHT, TEA
and SEA patties remained below this threshold during the whole
 J.M. Lorenzo et al. / Meat Science 96 (2014) 526–534
Table 2
Total colour difference (ΔE1 − 20) measured on the surface of raw patties between days 1
and 20 of chilled storage. Results are expressed as means ± standard error (n = 3).
ΔE1 − 20 SEM
Control 4.82 ± 0.84bc 0.59
BHT 5.11 ± 0.35bc 0.25
Tea 1.90 ± 0.30a 0.21
Chestnut 5.66 ± 0.46c 0.32
Seaweed 4.37 ± 0.16b 0.11
Grape 5.06 ± 0.01bc 0.38
Means with a different letter (a–c) within a column are significantly different (P b 0.05).
SEM: Standard error of the mean.
storage period, while TBAR'S values in CON and CHE patties went be-
yond this value by day 20.
Several authors have also reported antioxidant activity of other plant
extracts in raw meat patties (Bekhit et al., 2003; Ibrahim, Abou-Arab, &
Abu, 2010; Lara et al., 2011; Rodríguez-Carpena et al., 2011). Results
obtained with grape extract are in agreement with those reported by
Garrido et al. (2011), who observed an effect of a grape extract in the in-
hibition of lipid oxidation in pork patties. The lipophilic nature of grape
extract and BHT could explain its higher efficacy compared to chestnut
extract, which is water soluble, since the antioxidant active compounds
in grape extract and BHT would have dissolved in the lipid matrix of the
patties, and thus, could have decreased oxidation more directly. Other
studies have demonstrated that the addition of tea catechins
(200–400 mg/kg) has inhibitory effects on lipid oxidation in red meat
and poultry patties (Mitsumoto, O'Grady, Kerry, & Buckley, 2005).
According to these authors, this result could be explained due to the
ability of tea catechins to bind the iron atom of myoglobin, thus delaying
lipid oxidation by reacting with free radicals.
The GRA and TEA extracts were more effective against lipid oxidation
than SEA and CHE extracts, which is in accordance with results from
the in vitro antioxidant activity assays (Table 1). Consistently, extracts
from GRA and TEA presented higher concentrations of polyphenolic
compounds and more intensive antioxidant activity than SEA and CHE
extracts.
4. Conclusions
The addition of some natural antioxidant could have a preservative
effect in porcine patties stored under refrigeration. From a micro-
biological viewpoint, the use of tea extract could extend the shelf-life
of patties to 20 days of storage. Grape extract inhibited the
discolouration of refrigerated patties by reducing the loss of redness
and the increase of yellowness. This protective effect towards the desir-
able red colour of raw patties may influence consumers purchase deci-
sion. Therefore, tea and grape extracts are the most promising natural
compounds towards replacing the use of commercial antioxidants for
extending the shelf-life of porcine patties under refrigerated conditions.
Acknowledgements
Authors are grateful to Galician government (Xunta Galicia
09TAL006CT and 09MDS003CT projects) for the financial support. Special
thanks to ASOPORCEL (Lugo, Spain) for the pork samples supplied for this
research.
References
An, B. J., Kwak, J. H., Son, J. H., Park, J. M., Lee, J. Y., Jo, C., & Byun, M. W. (2004). Biological and
anti-microbial activity of irradiated green tea polyphenols. Food Chemistry, 88, 549–555.
Badr, H. M. (2007). Antioxidative activity of carnosine in gamma irradiated ground beef
and beef patties. Food Chemistry, 104, 665–679.
Barreira, J. C. M., Ferreira, I. C. F. R., Oliveira, M. B. P. P., & Pereira, J. A. (2008). Antioxidant
activities of the extracts from chestnut flower, leaf, skin and fruit. Food Chemistry, 107,
1106–1113.
533
Baydar, N. G., Ozkan, G., & Sagdic, O. (2004). Total phenolic contents and antibacterial
activities of grape (Vitis vinifera L.) extracts. Food Control, 15, 335–339.
Bekhit, A. E. D., Geesink, G. H., Ilian, M.A., Morton, J.D., & Bickerstaffe, R. (2003). The effects
of natural antioxidants on oxidative processes and metmyoglobin reducing activity in
beef patties. Food Chemistry, 81, 175–187.
Bonilla, F., Mayen, M., Merida, J., & Medina, M. (1999). Extraction of phenolic compounds
from red grape marc for use as food lipid antioxidants. Food Chemistry, 66, 209–215.
Bozan, B., Tosun, G., & Özcan, D. (2008). Study of polyphenol content in the seeds of red
grape (Vitis vinifera L.) varieties cultivated in Turkey and their antiradical activity.
Food Chemistry, 109, 426–430.
Brand-Williams, W., Cuvelier, M. E., & Berset, C. (1995). Use of a free radical method to
evaluate antioxidant activity. Lebensmittel-Wissenschaft und Technologie, 28, 25–30.
Carpenter, C. E., Cornforth, D. P., & Whittier, D. (2001). Consumer preference for beef
colour and packaging did not affect eating satisfaction. Meat Science, 57, 359–363.
Chouliara, E., Karatapanis, A., Savvaidis, I. N., & Kontominas, M. G. (2007). Combined effect
of oregano essential oil and modified atmosphere packaging on shelf-life extension of
fresh chicken breast meat, stored at 4 °C. Food Microbiology, 24, 607–617.
CIE (1978). International commission on illumination, recommendations on uniform colour
spaces, colour difference equations, psychometric colour terms. Supplement no. 15 to
CIE publication no. 15 (E-1.3.1) 1971/(TO-1.3)Paris, France: Bureau Central de la CIE.
Clausen, I., Jakobsen, M., Ertbjerg, P., & Madsen, N. T. (2009). Modified atmosphere
packaging affects lipid oxidation, myofibrillar fragmentation index and eating quality
of beef. Packaging Technology and Science, 22, 85–96.
Cruz, J. M., Dominguez, J. M., Dominguez, H., & Parajó, J. C. (2001). Antioxidant and anti-
microbial effects of extracts from hydrolysates of lignocellulosic materials. Journal of
Agricultural and Food Chemistry, 49, 2459–2464.
Dainty, R. H., & Mackey, B.M. (1992). The relationship between the phenotypic properties of
bacteria from chill-stored meat and spoilage processes. Journal of Applied Bacteriology, 73,
103–114.
Díaz-Reinoso, B., Moure, A., Domínguez, H., & Parajó, J. C. (2011). Membrane concentra-
tion of antioxidants from Castanea sativa leaves aqueous extracts. Chemical Engineer-
ing Journal, 175, 95–102.
Djenane, D., Sánchez-Escalante, A., Beltrán, J. A., & Roncalés, P. (2002). Ability of a-tocopherol,
taurine and rosemary, in combination with vitamin C, to increase the oxidative stability
of beef steaks packaged in modified atmosphere. Food Chemistry, 76, 407–415.
Estévez, M., Morcuende, D., & Cava, R. (2003). Oxidative and colour changes in meat from
three lines of free-range reared Iberian pigs slaughtered at 90 kg live weight and
from industrial pig during refrigerated storage. Meat Science, 65, 1139–1146.
Faustman, C., Sun, Q., Mancini, R., & Suman, S. P. (2010). Myoglobin and lipid oxidation
interactions: mechanistic bases and control. Meat Science, 86, 86–94.
Fontecave, M., Lepoivre, M., Elleingand, E., Gerez, C., & Guittet, O. (1998). Resveratrol, a re-
markable inhibitor of ribonucleotide reductase. FEBS Letters, 421, 277–279.
Francis, F. J., & Clydesdale, F. M. (1975). Food colorimetry: theory and applications. West-
port, CT: The AVI Publishing Co., Inc.
Garrido, M.D., Auqui, M., Martí, N., & Linares, M. B. (2011). Effect of two different red
grape pomace extracts obtained under different extraction systems on meat quality
of pork burgers. LWT — Food Science and Technology, 44, 2238–2243.
Georgantelis, D., Blekas, G., Katikou, P., Ambrosiadis, I., & Dimitiros, J. F. (2007). Effect of
rosemary extract, chitosan and α-tocopherol on lipid oxidation and colour stability
during frozen storage of beef burgers. Meat Science, 75, 266–274.
Greene, B. E., Hsin, I. M., & Zipser, M. W. (1971). Retardation of oxidative color changes in
raw ground beef. Journal of Food Science, 36, 940–942.
Haak, L., Raes, K., & De Smet, S. (2009). Effect of plant phenolic, tocopherol and ascorbic
acid on oxidative stability of pork patties. Journal of the Science of Food and Agriculture,
89, 1360–1365.
Hao, J. J., Liu, H., Donis-Gonzalez, I. R., Lu, X. H., Jones, D., & Fulbright, D. W. (2012). Anti-
microbial activity of chestnut extracts for potential use in managing soilborne plant
pathogens. Plant Disease, 96, 354–360.
Ibrahim, H. M., Abou-Arab, A. A., & Abu, S. F. M. (2010). Addition of some natural plant ex-
tracts and their effects on lamb patties quality. Journal of Food Technology, 8, 134–142.
Jakobsen, M., & Bertelsen, G. (2000). Colour stability and lipid oxidation of fresh beef.
Development of a response surface model for predicting the effects of temperature,
storage time, and modified atmosphere composition. Meat Science, 54, 49–57.
Jay, J. M., Loessner, M. J., & Golden, D. A. (2005). Modern food microbiology (7th ed.)New
York: Springer Science + Business Media.
Jayaprakasha, G. K., Selvi, T., & Sakariah, K. K. (2003). Antibacterial and antioxidant activ-
ities of grape (Vitis vinifera) seed extracts. Food Research International, 36, 117–122.
Juneja, V. K., Dwivedi, H. P., & Yan, X. (2012). Novel natural food antimicrobials. Annual
Review of Food Science and Technology, 3, 381–403.
Koutsoumanis, K. P., Stamatiou, A., Skandamis, P., & Nychas, G. -J. E. (2006). Development
of a microbial model for the combined effect of temperature and pH on spoilage of
ground meat and validation of the model under dynamic temperature conditions.
Applied and Environmental Microbiology, 72, 124–134.
Krzywicki, K. (1979). Assessment of relative content of myoglobin, oxymyoglobin and
metmyoglobin at the surface of beef. Meat Science, 3, 1–10.
Lara, M. S., Gutierrez, J. I., Timón, M., & Andrés, A. I. (2011). Evaluation of two natural
extracts (Rosmarinus officinalis L. and Melissa officinalis L.) as antioxidants in cooked
pork patties packed in MAP. Meat Science, 88, 481–488.
López-López, I., Bastida, S., Ruiz-Capillas, C., Bravo, L., Larrea, M. T., Sánchez-Muniz, F.,
Cofrades, S., & Jiménez-Colmenero, F. (2009). Composition and antioxidant capacity of
low-salt meat emulsion model systems containing edible seaweeds. Meat Science, 83,
492–498.
Lorenzo, J. M., & Gómez, M. (2012). Shelf life of fresh foal meat under MAP, overwrap and
vacuum packaging conditions. Meat Science, 92, 610–618.
Lotito, S. B., Actis-Goretta, L., Renart, M. -L., Caligiuri, M., Rein, D., Schmitz, H., Steinberg, F. M.,
Keen, C. L., & Fraga, C. (2000). Influence of oligomer chain length on the antioxidant
534 J.M. Lorenzo et al. / Meat Science 96 (2014) 526–534
activity of procyanidins. Biochemical and Biophysical Research Communications, 276,
945–951.
Lund, M. N., Lametsch, R., Hviid, M. S., Jensen, O. N., & Skibsted, L. H. (2007). High-oxygen
packaging atmosphere influences protein oxidation and tenderness of porcine
longissimus dorsi during chill storage. Meat Science, 77, 295–303.
Madhavi, D. L., & Salunkhe, D. K. (1995). Toxicological aspects of food antioxidants. In D. L.
Madavi, S. S. Deshpande, & D. K. Salunkhe (Eds.), Food antioxidants (pp. 267). New
York: Marcel Dekker.
Mandic, A. I., Dilas, S. M., Ćetković, G. S., Čanadanović-Brunet, J. M., & Tumbas, V. T. (2008).
Polyphenolic composition and antioxidant activities of grape seed extract. Interna-
tional Journal of Food Properties, 11, 713–726.
Marco, G. J. (1968). A rapid method for evaluation of antioxidants. Journal of the American
Oil Chemists' Society, 45, 594–598.
Mitsumoto, M., O'Grady, M. N., Kerry, J. P., & Buckley, D. J. (2005). Addition of tea catechins
and vitamin C on sensory evaluation, colour and lipid stability during chilled storage
in cooked or raw beef and chicken patties. Meat Science, 69, 773–779.
Perumalla, A. V. S., & Hettiarachchy, N. S. (2011). Green tea and grape seed extracts —
Potential applications in food safety and quality. Food Research International, 44, 827–839.
Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Rice-Evans, C. (1999).
Antioxidant activity applying an improved ABTS radical cation decolorization assay.
Free Radical Biology & Medicine, 26, 1231–1237.
Rial, D., Vázquez, J. A., & Murado, M. A. (2011). Effects of three heavy metals on the bac-
teria growth kinetics: A bivariate model for toxicological assessment. Applied Micro-
biology and Biotechnology, 90, 1095–1109.
Rockenbach, I. I., Silva, G. L., Rodrigues, E., Kuskoski, E. M., & Fett, R. (2008). Influência do
solvente no conteúdo total de polifenóis, antocianinas e atividade antioxidante de
extratos de bagaço de uva (Vitis vinifera) variedades Tannat e Ancelota. Ciência e
Tecnologia de Alimentos, 28, 238–244.
Rodríguez-Carpena, J. G., Morcuende, D., & Estévez, M. (2011). Avocado by-products as
inhibitors of colour deterioration and lipid and protein oxidation in raw porcine
patties subjected to chilled storage. Meat Science, 89, 166–173.
Rojas, M. C., & Brewer, S. (2008). Effect of natural antioxidants on oxidative stability of
frozen, vacuum-packaged beef and pork. Journal of Food Quality, 31, 173–188.
Rubilar, M., Pinelo, M., Shene, C., Sineiro, J., & Nuñez, M. J. (2007). Separation and HPLC–
MS identification of phenolic antioxidants from agricultural residues: Almond hulls
and grape pomace. Journal of Agricultural and Food Chemistry, 55, 10101–10109.
Saleem, M., Nazir, M., Ali, M. S., Hussain, H., Lee, Y. S., Riaz, N., & Jabbar, A. (2010). Antimi-
crobial natural products: An update on future antibiotic drug candidates. Natural
Product Reports, 27, 238–254.
Sánchez, M., Franco, D., Sineiro, J., Magariños, B., & Núñez, M. J. (2009). Antioxidant
power, bacteriostatic activity, and characterization of white grape pomace extracts
by HPLC-ESI-MS. European Food Research and Technology, 230, 291–301.
Santos, F. D., Rojas, M., Lockhorn, G., & Brewer, M. S. (2007). Effect of carbon monoxide in
modified atmosphere packaging, storage time and endpoint cooking temperature on
the internal color of enhanced pork. Meat Science, 77, 520–528.
Shanmughapriya, S., Manilal, A., Sujith, S., Selvin, J., Kiran, G. S., & Natarajaseenivasan, K.
(2008). Antimicrobial activity of seaweeds extracts against multiresistant pathogens.
Annals of Microbiology, 58, 535–541.
Singleton, V. L., Orthofer, R., & Lamuela-Raventós, R. M. (1999). Analysis of total phenols
and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent.
In L. Packer (Ed.), Methods in Enzymology, Oxidants and Antioxidants. Academic Press,
299. (pp. 152–178). CA: San Diego.
Tamil Selvi, A., Joseph, G. S., & Jayaprakasha, G. K. (2003). Inhibition of growth and afla-
toxin production in Aspergillus flavus by Garcinia indica extract and its antioxidant
activity. Food Microbiology, 20, 455–460.
Tesaki, S., Tanabe, S., Moriyama, M., Fukushi, E., Kawabata, J., & Watanabe, M. (1999). Iso-
lation and identification of an antibacterial compound from grape and its application
to foods. Nippon Nogeikagaku Kaishi, 73(2), 125–128.
Tourino, S., Selga, A., Jiménez, A., Juliá, L., Lozano, C., Lizárraga, D., Cascante, M., & Torres, J.
L. (2005). Procyanidin fractions from pine (Pinus pinaster) bark: Radical scavenging
power in solution, antioxidant activity in emulsion, and antiproliferative effect in mel-
anoma cells. Journal of Agricultural and Food Chemistry, 53, 4728–4735.
van der Hooft, J. J., Akermi, M., Ünlü, F. M., Mihaleva, V., Gomez-Roldan, V., Raoul, J., Bino,
R. J., de Vos, R. C. H., & Vervoort, J. (2012). Structural annotation and elucidation of
conjugated phenolic compounds in black, green, and white tea extracts. Journal of Ag-
ricultural and Food Chemistry, 60, 8841–8850.
Viljanen, K., Kylli, P., Kivikari, R., & Heinonen, M. (2004). Inhibition of protein and lipid ox-
idation in liposomes by berry phenolics. Journal of Agricultural and Food Chemistry, 52,
7419–7424.
Vyncke, W. (1975). Evaluation of the direct thiobarbituric acid extraction method for de-
termining oxidative rancidity in mackerel (Scomber scombrus L.). Fette seifen
Anstichm, 77, 239–240.